During inflammation, flowing leukocytes roll on vascular surfaces through interactions of selectins with their glycosylated ligands. Activated endothelial cells express P-selectin and E-selectin. Their major ligands on leukocytes are P-selectin glycoprotein ligand-1 (PSGL-1), which binds to P- and E-selectin, and CD44, which binds to E-selectin. Data obtained during the previous funding period demonstrated that the organization of selectins and their ligands on cell surfaces has major impact on functions. We hypothesize that selectins and their ligands form homodimers through self-associations of their transmembrane domains. They localize in specialized membrane regions (e.g. microvilli, clathrin-coated pits, or lipid rafts) through interactions of their transmembrane or cytoplasmic domains with lipids, adaptor proteins, or cytoskeletal elements. Dimerization and membrane-domain targeting cooperate to enhance adhesive and signaling functions. We propose to test these hypotheses by making monomeric and dimeric forms of P-selectin, E-selectin, PSGL-1, and CD44 that do or do not target to lipid rafts or clathrin-coated pits. Other variants will test interactions with cytoskeletal and signaling proteins. Cells from knockout mice lacking signaling components will also be used. These tools will probe how cells organize selectins and their ligands to regulate leukocyte adhesion and signaling in vitro and in vivo. Because selectins and their ligands are major contributors to pathological inflammation and thrombosis, understanding how they function in their cellular environments may suggest new opportunities for therapeutic intervention. PUBLIC HEALTH RELEVANCE: White blood cells (leukocytes) combat infection and repair tissue injury. To do this, they must move from blood into the affected tissues, a process that begins when circulating leukocytes attach to blood vessel surfaces through adhesion receptors. This project addresses how leukocytes and cells lining blood vessels control the display and function of these receptors. Because excessive recruitment of leukocytes contributes to many diseases, including heart attacks and strokes, understanding how cells regulate adhesion receptors may suggest new therapeutic strategies.